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Journal of Applied Phycology

, Volume 31, Issue 2, pp 1163–1174 | Cite as

An optimized method and a dominant selectable marker for genetic engineering of an industrially promising microalga—Pavlova lutheri

  • Binod PrasadEmail author
  • Wolfgang Lein
  • Christoph Peter Lindenberger
  • Rainer Buchholz
  • Nithya Vadakedath
Article

Abstract

The marine microalga Pavlova lutheri has great nutritional value as it synthesizes and accumulates higher amounts of polyunsaturated fatty acids (PUFAs). It is commonly used in aquaculture. However, no transformation procedure has been realized for this industrially important microalga so far. Here, we report an efficient protocol for the nuclear transformation of P. lutheri. Agrobacterium-mediated transformation (AMT) of P. lutheri with a mutated genomic clone of phytoene desaturase (pds) gene, pds-L504R, from Haematococcus pluvialis yielded norflurazon-resistant P. lutheri cells. Ideal co-cultivation conditions for achieving higher numbers of transformants was found to be artificial seawater (ASW) medium, 100 μM acetosyringone, and a 24-h co-cultivation at 25 ± 1 °C. The integration of the introduced gene into the nuclear genome of P. lutheri was shown by PCR amplification of the T-DNA sequences from the genomic DNA of transformants and Southern blot analysis using T-DNA sequences as probes. The transgene expressed efficiently as evidenced by the results of stability and tolerance study, and the qRT-PCR analysis. Results clearly demonstrate the application of AMT approach and pds gene as a dominant selectable marker for the genetic engineering of P. lutheri for fundamental studies and biotechnological applications.

Keywords

Pavlova lutheri Agrobacterium tumefaciens Transformation Norflurazon Phytoene desaturase 

Notes

Acknowledgments

The authors are thankful to Dr. J. Steinbrenner (Universität Konstanz, Germany) for kindly providing the plasmid pPLAT-pds-L504R. The authors appreciate the Korea Institute of Advanced Technology, Korea and Federal Ministry of Education and Research, Germany for supporting the work. BP also acknowledges Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-University of Erlangen Nuremberg, Germany for the research support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10811_2018_1617_MOESM1_ESM.docx (602 kb)
ESM 1 (DOCX 601 kb)

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Binod Prasad
    • 1
    Email author
  • Wolfgang Lein
    • 2
    • 3
  • Christoph Peter Lindenberger
    • 4
  • Rainer Buchholz
    • 1
  • Nithya Vadakedath
    • 1
    • 3
  1. 1.Institute of Bioprocess EngineeringFriedrich-Alexander University Erlangen-NürnbergErlangenGermany
  2. 2.Institute for BiotechnologyTechnical University BerlinBerlinGermany
  3. 3.Department of BiotechnologyDongseo UniversityBusanSouth Korea
  4. 4.Institute of Bioprocess EngineeringFriedrich-Alexander Universität Erlangen-Nürnberg Campus BusanBusanSouth Korea

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